Ligand/lytic peptide methods of use

ABSTRACT

Amphipathic lytic peptides are ideally suited to use in a ligand/cytotoxin combination to specifically inhibit cells that are driven by or are dependent upon a specific ligand interaction; for example, to induce sterility or long-term contraception, or to attack tumor cells, or to selectively lyse virally-infected cells, or to attack lymphocytes responsible for autoimmune diseases. The peptides act directly on cell membranes, and need not be internalized. Administering a combination of gonadotropin-releasing hormone (GnRH) (or a GnRH agonist) and a membrane-active lytic peptide produces long-term contraception or sterilization in animals in vivo. Administering in vivo a combination of a ligand and a membrane-active lytic peptide kills cells with a receptor for the ligand. The compounds are relatively small, and are not antigenic. Lysis of gonadotropes has been observed to be very rapid (on the order of ten minutes.) Lysis of tumor cells is rapid. The two components—the ligand and the lytic peptide—may optionally be administered as a fusion peptide, or they may be administered separately, with the ligand administered slightly before the lytic peptide, to activate cells with receptors for the ligand, and thereby make those cells susceptible to lysis by the lytic peptide. The compounds may be used in gene therapy to treat malignant or non-malignant tumors, and other diseases caused by clones or populations of “normal” host cells bearing specific receptors (such as lymphocytes), because genes encoding a lytic peptide or encoding a lytic peptide/peptide hormone fusion may readily be inserted into hematopoietic stem cells or myeloid precursor cells.

RELATED APPLICATIONS

This application is a divisional of application Ser. No. 10/617,561,filed Jul. 11, 2003 now U.S. Pat. No. 7,566,777, which is a continuationof application Ser. No. 09/381,879, filed Sep. 24, 1999 (now U.S. Pat.No. 6,635,740), which is a national phase application of internationalapplication no. PCT/US98/06114, filed Mar. 27, 1998, which claimspriority to provisional application Ser. No. 60/041,009, filed Mar. 27,1997; application Ser. No. 09/869,153, filed Jun. 4, 1997 now convertedto provisional application Ser. No. 60/092,112, and provisionalapplication Ser. No. 60/057,456, filed Sep. 3, 1997. All of theforegoing applications identified in this paragraph are incorporatedherein by reference in their entirety.

TECHNICAL FIELD

This invention pertains to compositions and methods for specificallyinhibiting cells that are driven by or are dependent on specific ligandinteractions. Examples are compositions and methods for long-termcontraception or sterilization; compositions and methods for inhibitingor killing malignant and non-malignant, hormone-dependent tumors;compositions and methods for selectively killing virally infected cells;and compositions and methods for selectively destroying lymphocytesresponsible for autoimmune disorders.

BACKGROUND ART

Compositions that have sometimes been used for long-term contraceptioninclude those based upon natural or synthetic steroidal hormones to“trick” the female reproductive tract into a “false pregnancy.” Thesesteroidal hormones must be administered repeatedly to prevent completionof the estrous cycle and conception. Steroids have side effects that canbe potentially dangerous.

P. Olson et al., “Endocrine Regulation of the Corpus Luteum of the Bitchas a Potential Target for Altering Fertility,” J. Reprod. Fert. Suppl.,vol. 39, pp. 27-40 (1989) discusses the luteal phase and its regulationin bitches. The following discussion appears at page 37: “Specifictoxins can be linked to an antibody or hormone and carried to a specifictarget cell (or cells) which is then killed by the toxin. The idea ofdeveloping a ‘magic bullet’ has been discussed for decades but is nowgaining renewed recognition as a potential, highly selective method fordestroying specific tissues while leaving other tissues unharmed. Formany years it was impossible to develop large quantities of antibodieswhich would react specifically with only single antigenic determinants.However, with the advent of monoclonal antibodies, this problem has beenlargely overcome. Antibodies can be developed to specific hormonereceptors (such as the LH receptor) and then coupled to a toxin. Allcells with LH receptors should then be destroyed. Although various celltypes have not been characterized in dog corpora lutea, destruction ofany luteal cell type could potentially result in luteolysis if celltypes communicate.” (citations omitted)

P. Olson et al., “New Developments in Small Animal Population Control,”JAVMA, vol. 202, pp. 904-909 (1993) gives an overview of methods forpreventing or terminating unwanted pregnancies in small animals. Thefollowing discussion appears at page 905: “Tissue-specificcytotoxins—Permanent contraception in females and males might beachieved by administration of a cytotoxin that is linked togonadotropin-releasing hormone (GnRH) and that selectively destroysgonadotropin-secreting pituitary cells. Similarly, a cytotoxin linked toantibodies against gonadotropin receptors could be targeted to altergonadal function. Toxins would need to be carefully targeted to specificcells, yet be safe for all other body tissues.” (citation omitted).

T. Janaky et al., “Short Chain Analogs of Luteinizing Hormone-ReleasingHormone Containing Cytotoxic Moieties,” Proc. Natl. Acad. Sci. USA, vol.89, pp. 10203-10207 (1992) discloses the use of certain hexapeptide andheptapeptide analogs of GnRH as carriers for certain alkylating nitrogenmustards, certain anthraquinone derivatives, antimetabolite, andcisplatin-like platinum complex. The authors reported that several ofthe compounds exerted some cytotoxic effects on the MCF-7 breast cancercell line.

D. Fitzgerald et al., “Targeted Toxin Therapy for the Treatment ofCancer,” J. Natl. Cancer Inst., vol. 81, pp. 1455-1463 (1989), reviewedtargeted toxin therapies for cancers, including conjugating toxins suchas Pseudomonas exotoxin, diphtheria toxin, and ricin to a cell-bindingprotein such as a monoclonal antibody or a growth factor. The conjugatesare then internalized into cytoplasm, where the toxin disrupts cellularactivity.

Conventional targeted toxin therapies have several drawbacks. There is asmall window for treatment with a particular targeted toxin (on theorder of two weeks) before the recipients immune system mounts anantibody response to the targeted toxin. These antibodies willneutralize the toxin; or worse, may result in deposition of the toxin inreticuloendothelial tissues (e.g., liver, spleen, lymph nodes, lungs,bone marrow), where they may damage otherwise healthy tissue. Aside fromthis drawback, the toxin must be internalized by the targeted cell andtranslocated into the cytoplasm to have effect.

A related approach is to link a monoclonal antibody to an enzyme. Thisconjugate is directed specifically to a tumor cell surface antigen. Aprodrug is then administered to the patient. The prodrug issubstantially less toxic than the drug that results from activation ofthe drug at the tumor site by the conjugated enzyme. The activated drugthen selectively attacks tumor cells. See, e.g., D. Kerr et al.,“Regressions and Cures of Melanoma Xenografts following Treatment withMonoclonal Antibody β-Lactamase Conjugates in Combination withAnticancer Prodrugs,” Cancer Research, vol. 55, pp. 3558-3563 (1995);and H. Svensson et al., “In vitro and In Vivo Activities of aDoxorubicin Prodrug in Combination with Monoclonal Antibody β-LactamaseConjugates,” Cancer Research, vol. 55, pp. 2357-2365 (1995).

S. Sealfon et al., “Molecular mechanisms of ligand interaction with thegonadotropin-releasing hormone receptor,” Endocrine Reviews, vol. 18,pp. 180-205 (1997) provides a review of research concerning theinteraction between GnRH and its receptor.

F. Hu et al., “Theophylline and Melanocyte-Stimulating Hormone Effectson Gamma-Glutamyl Transpeptidase and DOPA Reactions in Cultured MelanomaCells,” J. Investigative Dermatology, vol. 79, pp. 57-61 (1982)disclosed that theophylline and melanocyte-stimulating hormone (MSH)both enhanced pigmentation in murine melanoma cells, apparently bydifferent mechanisms. J. Murphy et al., “Genetic Construction,Expression, and Melanoma-Selective Cytotoxicity of a DiphtheriaToxin-Related α-Melanocyte-Stimulating Hormone Fusion Peptide,” Proc.Natl. Acad. Sci. USA, vol. 83, pp. 8258-8262 (1986) discloses selectiveactivity against melanoma cells in vitro by an MSH-diphtheria toxinconjugate. See also D. Bard, “An Improved Imaging Agent for MalignantMelanoma, Based on [Nle⁴, D-Phe⁷]α-Melanocyte Stimulating Hormone,”Nucl. Med. Comm., vol. 16, pp. 860-866 (1995).

W. Siegrist et al., “Homologous and Heterologous Regulation ofα-Melanocyte-Stimulating Hormone Receptors in Human and Mouse MelanomaCell Lines,” Cancer Research, vol. 54, pp. 2604-2610 (1994) reports thatit is well-established that human melanoma cells possess specific highaffinity receptors for α-MSH. See also J. Tatro et al., “MelanotropinReceptors Demonstrated In Situ in Human Melanoma,” J. Clin. Invest.,vol. 85, pp. 1825-1832 (1990).

P. Bacha et al., “Thyrotropin-Releasing Hormone-Diphtheria Toxin-relatedPolypeptide Conjugates,” J. Biol. Chem., vol. 258, pp. 1565-1570 (1983)discloses conjugates of thyrotropin-releasing hormone (TRH) with twodiphtheria toxins; one of these conjugates caused a 50% inhibition ofprotein synthesis in rat GH₃ pituitary cells at 3×10⁻⁹ M concentration.See also P. Bacha et al., “Organ-Specific Binding of aThyrotropin-Releasing Hormone-Diphtheria Toxin Complex after IntravenousAdministration to Rats,” Endocrinology, vol. 113, pp. 1072-1076 (1983).

V. Chaudhary, “Activity of a Recombinant Fusion Protein betweenTransforming Growth Factor Type α and Pseudomonas toxin,” Proc. Natl.Acad. Sci. USA, vol. 84, pp. 4538-4542 (1987) discloses that a fusionprotein of a modified Pseudomonas toxin and transforming growth factortype a selectively kills cells expressing epidermal growth factorreceptors. See also D. Cawley et al., “Epidermal Growth Factor-Toxin AChain Conjugates: EGF-Ricin Is a Potent Toxin while EGF-DiphtheriaFragment A is Nontoxic,” Cell, vol. 22, pp. 563-570 (1980).

E. Vitetta et al., “Redesigning Nature's Poisons to Create Anti-TumorReagents,” Science, vol. 238, pp. 1098-1104 (1987) reviews the use ofimmunotoxins against tumors. Uses in preventing graft-versus-hostreactions are also mentioned. The authors mentioned that in vivoeffectiveness was less than desirable. Difficulties mentioned includedaccessibility of toxins in circulation to target cells; instability ofthe linkage of toxin to antibody; rapid clearance of the immunotoxinsfrom circulation by the liver; response by the recipient's immune systemto the toxin or to the monoclonal antibody, complicating long-termtherapy; possible lack of specificity for neoplastic renewal cells;cross-reactivity with normal cells; heterogeneity of tumor cells; andshedding of surface antigens by tumor cells.

P. Trail et al., “Antigen-specific Activity of Carcinoma-reactiveBR64-Doxorubicin Conjugates Evaluated in Vitro and in Human TumorXenograft Models,” Cancer Research, vol. 52, pp. 5693-5700 (1992)disclose the conjugation of the anticarcinoma antibody BR64 to adoxorubicin derivative, and discuss the antitumor effects of theconjugate.

J. Olson, “Laboratory Evidence for the Hormonal Dependency ofMeningiomas,” Human Reproduction, vol. 9, supp. 1, pp. 195-201 (1994)discloses evidence that meningiomas, benign intracranial tumors, possessprogesterone receptors.

S. Prigent et al., “The Type 1 (EGFR-Related) Family of Growth FactorReceptors and their Ligands,” Progress in Growth Factor Research, vol.4, pp. 1-24 (1992) reviews the biology of the epidermal growth factor(EGF), its receptor, and related ligands and receptors (e.g., c-erbB-2,c-erbB-3, TGFα, amphiregulin, heregulin), and their roles in normal cellproliferation and in the pathogenesis of human cancer. See also D.Davies et al., “Targeting the Epidermal Growth Factor Receptor forTherapy of Carcinomas,” Biochem. Pharm., vol. 51, pp. 1101-1110 (1996).

D. Morbeck et al., “A Receptor Binding Site Identified in the Region81-95 of the β-Subunit of Human Luteinizing Hormone (LH) and chorionicgonadotropin (hCG),” Molecular and Cellular Endocrinology, vol. 97, pp.173-181 (1993) disclosed a fifteen amino acid region of LH and hCG thatacted as a receptor binding site. (LH and hCG are homologous hormonesthat produce similar effects.)

W. Theunis et al., “Luteinising Hormone, Follicle Stimulating Hormoneand Gonadotropin Releasing Hormone Immunoreactivity in Two Insects:Locusta migratoria migratoroides R & F and Sarcophaga bullata (Parker),”Invert. Reprod. and Develop., vol. 16, pp. 111-117 (1989) disclosed thatmaterials immunologically related to LH, FSH, and GnRH were localized incerebral tissue of Locusta migratoria and Sarcophaga bullata. See alsoP. Verhaert et al., “Substances Resembling Peptides of the VertebrateGonadotropin System Occur in the Central Nervous System of Periplanetaamericana L.,” Insect Biochem., vol. 16, pp. 191-197 (1986).

U.S. Pat. Nos. 5,378,688; 5,488,036; and 5,492,893 disclose compoundssaid to be useful in inducing sterility in mammals, and in treatingcertain sex hormone-related cancers in mammals. The disclosed compoundswere generically described as GnRH (or a GnRH analog) conjugated to atoxin. The toxin was preferably linked to the sixth amino acid of theGnRH agonist. The toxin was preferably one with a translocation domainto facilitate uptake into a cell. The inventors noted that conjugationof the GnRH agonist to the toxin “is necessary because, for the mostpart, the above toxins, by themselves, are not capable of binding withcell membranes in general. That is to say that applicants have foundthat it is only when a GnRH analog of the type described herein islinked to a toxin of the type noted above does that toxin become capableof binding to cell membranes . . . .” (E.g., U.S. Pat. No. 5,488,036,col. 7, lines 46-52.) The toxins specifically mentioned appear all tohave been metabolic toxins, for example ricin, abrin, modeccin, variousplant-derived ribosome-inhibiting proteins, pokeweed antiviral protein,α-amanitin, diphtheria toxin, pseudomonas exotoxin, shiga toxin,melphalan, methotrexate, nitrogen mustard, doxorubicin, and daunomycin.None of these toxins is believed to be toxic due to direct interactionwith the cell membrane. In the in vivo experiments reported, the mosteffective time course was reported to be weekly injections for 4 weeks.(E.g., U.S. Pat. No. 5,488,036, col. 20, lines 46-47.) Because most ofthe conjugates cited are relatively large compounds, antigenicity couldbe a problem when such multiple administrations are used. The GnRHanalog was preferably linked to the toxin with one of several specifiedheterobifunctional reagents. The specifications suggest thatconsiderable effort was expended in conjugating the toxin to the GnRHagonist. The toxins must in general be internalized into the targetcells to have effect, and do not act on cell membranes; in addition, atleast some of these toxins must be secondarily transported from themembrane-bound vesicle into the cytoplasm to interact with ribosomes,mitochondria, or other cellular components.

M. Kovacs et al., “Recovery of pituitary function after treatment with atargeted cytotoxic analog of luteinizing hormone-releasing hormone,”Proc. Natl. Acad. Sci. USA, vol. 94, pp. 1420-1425 (1997) discloses thata doxorubin analog conjugated to an LH-RH (i.e., GnRH) agonistselectively attacked cells with LH-RH receptors, and that its effect onpituitary cells was reversible. The paper suggests that the conjugatemight be used to treat tumors with LH-RH receptors. See also A.Jungwirth et al., “Regression of rat Dunning R-3227-H prostate carcinomaby treatment with targeted cytotoxic analog of luteinizinghormone-releasing hormone AN-207 containing 2-pyrrolinodoxorubicin,”Intl. J. Oncol., vol. 10, pp. 877-884 (1997)

R. Moretti et al., “Luteinizing hormone-releasing hormone agonistsinterfere with the stimulatory actions of epidermal growth factor inhuman prostatic cancer cell lines, LNCaP and DU 145,” J. Clin. Endocrin.& Metab., vol. 81, pp. 3930-3937 (1996) discloses that LH-releasinghormone agonists inhibit both androgen-dependent (LNCaP) andandrogen-independent (DU 145) human prostatic cancer cell lines, andsuggests that the agonists may inhibit proliferation of the tumor cellsby interfering with the stimulatory actions of epidermal growth factor.

I. Mezô et al., “Synthesis of GnRH analogs having direct antitumor andlow LH-releasing activity,” J. Med. Chem., vol. 40, pp. 3353-3358 (1997)discloses chicken I GnRH agonists and antagonists. Agonist MI-1892 wasreported to have low endocrinological activity, but to possess antitumoractivity.

A. Nechushtan et al., “Adenocarcinoma cells are targeted by the newGnRH-PE₆₆ chimeric toxin through specific gonadotropin-releasing hormonebinding sites,” J. Biol. Chem., vol. 272, pp. 11597-11603 (1997)discloses the use of a Pseudomonas exotoxin coupled to GnRH to killcertain types of cancer cells.

X. Zhu, “Steroid-independent activation of androgen receptor inandrogen-independent prostate cancer. A possible role for the MAP kinasesignal transduction pathway?” Mol. & Cell. Endocrinol., vol. 134, pp.9-14 (1997) discloses that androgen receptors in prostate cancer couldbe activated in the absence of the androgen signal.

G. Emons et al., “Growth-inhibitory actions of analogues of luteinizinghormone releasing hormone on tumor cells,” Trends in Endocrin. Metab.,vol. 8, pp. 355-362 (1997) reviews the similarities and differencesbetween GnRH receptors of cancer cells and of normal brain and pituitarycells; and suggests that LHRH analogs interfere with the mitogenicsignal transduction of growth-factor receptors and related oncogeneproducts associated with tyrosine kinase activity in a number ofmalignant human tumors, including breast, ovary, endometrium, andprostate cancers.

D. Tang et al., “Target to Apoptosis: A Hopeful Weapon for ProstateCancer,” The Prostate, vol. 32, pp. 284-293 (1997) provides a review ofresearch on apoptosis as a route to treat prostate cancers.

A. Goustin et al., “Growth Factors and Cancer,” Cancer Research, vol.46, pp. 1015-1029 (1986) provides an overview of various growth factorsthat have been associated with different cancers.

S. Cho et al., “Evidence for autocrine inhibition ofgonadotropin-releasing hormone (GnRH) gene transcription by GnRH inhypothalamic GT1-1 neuronal cells,” Mol. Brain Res., vol. 50, pp. 51-58(1997) discloses that neuroendocrine populations of GnRH neurons havehigh affinity receptors for GnRH and for GnRH analogs.

S. Sower et al., “Primary structure and biological activity of a thirdgonadotropin-releasing hormone from lamprey brain,” Endocrinology, vol.132, pp. 1125-1131 (1993) describes the structure of lamprey III GnRH.

E. Stopa et al., “Immunocytochemical evidence for a lamprey-likegonadotropin-releasing hormone in human brain,” Soc. Neurosci. Abstr.,abstract no. 437.8, p. 1577 (1987) discloses that a lamprey-like GnRHIII is found in humans.

S. White et al., “Three gonadotropin-releasing hormone genes in oneorganism suggest novel roles for an ancient peptide,” Proc. Natl. Acad.Sci. USA, vol. 92, pp. 8363-8367 (1995); and J. Powell et al., “Threeforms of gonadotropin-releasing hormone characterized from brains of onespecies,” Proc. Natl. Acad. Sci. USA, vol. 91, pp. 12081-12085 (1994)are examples of papers reporting the typical presence of three forms ofGnRH in species of vertebrates.

J. Wamock et al., “Anxiety and mood disorders associated withgonadotropin-releasing hormone agonist therapy,” PsychopharmacologyBull., vol. 33, pp. 311-316 (1997) reports that psychological sideeffects can accompany chronic treatment with a GnRH agonist.

L. Deligdisch et al., “Pathological changes in gonadotropin releasinghormone agonist analogue treated uterine leiomyomata,” Fertility andSterility, vol. 67, pp. 837-841 reported the pathological changesassociated with treating leiomyomata with a GnRH analog to induceiatrogenic menopause.

J. Fuerst et al., “Effect of active immunization against luteinizinghormone-releasing hormone on the androgen-sensitive Dunning R3327-PAPand Androgen-Independent Dunning R3327-AT2.1 prostate cancer sublines,”Prostate, vol. 32, pp. 77-84 (1997) reported that active immunization ofrats with an LHRH-diphtheria toxoid conjugate caused atrophy of thetestes, prostate, and androgen-sensitive prostate tumors, withinhibition of the tumors caused by suppression of cell division ratherthan an increase in cell death; and that the volume increase ofandrogen-independent prostate tumors was slightly reduced.

C. Mantzoros et al., “Insulin-like growth factor 1 in relation toprostate cancer and benign prostatic hyperplasia,” Br. J. Cancer, vol.76, pp. 1115-1118 (1997) reported that increased levels of insulin-likegrowth factor 1 were associated with an increased risk of prostatecancer.

V. Ding, “Sex hormone-binding globulin mediates prostate androgenreceptor action via a novel signaling pathway,” Endocrinology, vol. 139,pp. 213-218 (1998) reported that androgen-independent pathways mayactivate the progression of some prostate cancers.

J. King et al., “Evolution of gonadotropin-releasing hormones,” Trendsin Endocrin. Metab., vol. 3, pp. 339-344 (1992) discloses the primarystructures of different GnRHs from various vertebrates. See also J. Kinget al., “Structure of chicken hypothalamic luteinizing hormone-releasinghormone. II. Isolation and characterization,” J. Biol. Chem., vol. 257,pp. 10729-10732 (1982).

N. Mores et al., “Activation of LH receptors expressed in GnRH neuronsstimulates cyclic AMP production and inhibits pulsatile neuropeptiderelease,” Endocrinology, vol. 137, pp. 5731-5734 (1996) discloses thatLH acts directly on neuroendocrine neurons in the brain. See also Z. Leiet al., “Signaling and transacting factors in the transcriptionalinhibition of gonadotropin releasing hormone gene by human chorionicgonadotropin in immortalized hypothalamic GT1-7 neurons,” Mol. & Cell.Endocrinology, vol. 109, pp. 151-157 (1995).

U.S. Pat. Nos. 5,597,945 and 5,597,946 disclose plants transformed withgenes encoding various lytic peptides.

DISCLOSURE OF INVENTION

It has been unexpectedly discovered that amphipathic lytic peptides areideally suited to use in a ligand/cytotoxin combination to specificallyinhibit abnormal or normal cells that are driven by or are dependentupon a specific ligand interaction; for example, to induce sterility orlong-term contraception, or to attack tumor cells, or to selectivelylyse virally-infected cells, or to attack lymphocytes responsible forautoimmune diseases. The peptides act directly on cell membranes, andneed not be internalized.

For example, administering a combination of gonadotropin-releasinghormone (GnRH) (or a GnRH agonist) and a membrane-active lytic peptideproduces long-term contraception or sterilization in animals in vivo.Particularly surprising, sterility results even when the combination isadministered to a sexually immature animal: The combination thenprevents sexual maturation.

Administering in vivo a combination of a ligand and a membrane-activelytic peptide kills cells with a receptor for the ligand. The compoundsused in the present invention are relatively small, and will not beantigenic. (Lytic peptides are known not to be very antigenic; and theligands are not antigenic at all.) The compounds may be administered ina single dose, or in two or more closely spaced doses. Lysis ofgonadotropes has been observed to be very rapid (on the order of tenminutes.) Lysis of tumor cells is rapid. The two components—the ligandand the lytic peptide—may optionally be administered as a fusionpeptide, or they may be administered separately, with the ligandadministered slightly before the lytic peptide, to activate cells withreceptors for the ligand, and thereby make those cells susceptible tolysis by the lytic peptide. If a fusion peptide is used, it has beenunexpectedly discovered that a linking moiety is not necessary to jointhe ligand to the lytic peptide: one may be bonded directly to theother, without the need for any intervening linkage; bonding may beperformed by bonding one end of the ligand to one end of the peptide, orby bonding to the middle of either. The toxin, the lytic peptide, doesnot need a translocation domain, and need not be internalized, as itbinds to and acts directly on the activated cell membrane to causelysis. The ligand may be a full native compound, or it may instead bethe binding domain alone; the latter is preferred where the full ligandis relatively large.

The compounds of the present invention are well-suited for use in genetherapy to treat malignant or non-malignant tumors, and other diseasescaused by clones or populations of “normal” host cells bearing specificreceptors (such as lymphocytes), because genes encoding a lytic peptideor encoding a lytic peptide/peptide hormone fusion may readily beinserted into hematopoietic stem cells or myeloid precursor cells.

MODES FOR CARRYING OUT THE INVENTION

Several cancer cells (uterine, endometrial, prostate, testicular, andovarian) express LH or hCG receptors. Tao et al., “Expression ofLuteinizing Hormone/Human Chorionic Gonadotropin Receptor Gene in BenignProstatic Hyperplasia and in Prostatic Carcinoma in Humans,” Biol.Reprod., vol. 56, pp. 67-72 (1997). Conjugates of a lytic peptide and LHor a portion of the LH molecule may thus be used to destroy these cellsselectively. For example, the genes encoding such hormones as FSH, TRH,and LH are known, and may be linked to a DNA sequence encoding a lyticpeptide to produce a secreted fusion peptide, all under the control of asuitable promoter such as the acute-phase responsive promoters disclosedin U.S. patent application Ser. No. 08/474,678, filed Jun. 7, 1995, andin PCT application WO 95/01095, published Jan. 12, 1995. A binding sitefrom a hormone may be used in lieu of the entire hormone, for examplethe fifteen amino acid binding site of LH and hCG. See D. Morbeck etal., “A Receptor Binding Site Identified in the Region 81-95 of theβ-Subunit of Human Luteinizing Hormone (LH) and chorionic gonadotropin(hCG),” Molecular and Cellular Endocrinology, vol. 97, pp. 173-181(1993).

A powerful vector that is suitable for transforming cells to be used ingene therapy is the transposon-based vector that is disclosed in U.S.Pat. No. 5,719,055.

It is known that the D-amino acid form of GnRH will bind to gonadotropesin the pituitary, to GnRH neurons in the brain, and to various types ofcancer cells. It is also known that the D-amino acid forms of lyticpeptides have essentially the same propensity to lyse cell membranes asdo the L-amino acid forms. Compounds of the present invention (whetheradministered as a fusion peptide or separately) may therefore beadministered either in L-form or D-form. D-form peptides, althoughgenerally more expensive than L-form, have the advantage that they arenot degraded by normal enzymatic processes, so that the D-form peptidesmay therefore be administered orally and generally have a longerbiological half-life. Oral administration of the D-form peptide may beenhanced by linking the peptide/hormone fusion product to a suitablecarrier to facilitate uptake by the intestine, for example vitamin B₁₂,following generally the B₁₂-conjugation technique of G. Russell-Jones etal., “Synthesis of LHRH Antagonists Suitable for Oral Administration viathe Vitamin B₁₂ Uptake System,” Bioconjugate Chem., vol. 6, pp. 34-42(1995).

GnRH or GnRH analogs (collectively, “GnRH agonists”) may be used in thepresent invention. It has been reported that substitutions at the 6 and10 positions of the GnRH decapeptide can produce “superagonists” havinggreater binding affinity to the GnRH receptor than does GnRH itself.These “superagonists” include goserelin, leuprolide, buserelin, andnafarelin. See U.S. Pat. No. 5,488,036.

Without wishing to be bound by this theory, it is believed that amechanism (though not the exclusive mechanism) underlying thesterilization/long term contraception aspect of this invention is asfollows: GnRH activates gonadotropic cells in the pituitary gland, aswell as neuroendocrine GnRH neurons in the brain. The activated cellshave substantially increased susceptibility to lysis by a lytic peptide.The lytic peptide then preferentially destroys (or severely damages)these activated cells. When the gonadotrophic cells in the pituitary aredestroyed and are deprived of GnRH from the brain, the pituitary nolonger secretes follicle stimulating hormone (FSH) or luteinizinghormone (LH), rendering the animal temporarily or permanently sterile.

Although the ligand and the lytic peptide may be administeredseparately, it is preferred to link the two in a single molecule,because such a linkage greatly increases the effective concentration ofthe lytic peptide in the vicinity of ligand-activated cells.Furthermore, this increase in the effective lytic peptide concentrationcan obviate the need for activation of the cells, allowing the peptideto be linked to a binding site of a ligand alone, without needing toinclude the “remainder” of a native ligand that would normally be neededfor activating the target cells. This linkage may be in either order:for example, GnRH/peptide or peptide/GnRH. Examples are modifiedGnRH/hecate (SEQ. ID NO. 3) and hecate/modified GnRH (SEQ. ID NO. 4).Note that no intermediate linker is necessary, and that the carboxyterminus of one of the two peptides may be bonded directly to the aminoterminus of the other. (We have found that the initial pyro-glutamicacid residue of GnRH or of the GnRH portion of a fusion peptide may besubstituted with glutamine without substantially changing the activityof the respective peptides. See, e.g., SEQ. ID Nos. 9, 3, and 4.)

Experimental Results

EXAMPLES 1-6

The pituitary gland of an adult female rat was harvested and dividedinto six sections of approximately equal size. One section was placed ineach of six wells containing tissue culture medium at 37° C. A differenttreatment was applied to each well, as described below. Ten hours aftertreatment, the tissue from each well was fixed, and the histology ofeach was examined microscopically.

Treatment 1 applied tissue culture medium alone as a control. Thehistology of this tissue after treatment appeared normal.

Treatment 2 was an application of 5 nanograms of GnRH (SEQ. ID NO. 1)per mL of medium. The histology of this tissue after treatment wasnormal; a small degree of cellular vacuolization was noted. Forcomparison, the concentration of GnRH in normal, untreated rats variesfrom as low as 1 ng/mL to as high as 20 ng/mL during the LH surge phaseof the estrous cycle.

Treatment 3 was an application of 50 μM of the lytic peptide hecate(SEQ. ID NO. 2) in the medium. The histology of this tissue aftertreatment appeared normal.

Treatment 4 was an initial application of 5 nanograms of GnRH per mL ofmedium for 15 minutes. Following this incubation, the medium containingGnRH was removed, and the tissue was washed once with plain medium. Thismedium was then removed, and was replaced with medium containing 50 μMof the lytic peptide hecate. Widespread basophilic (gonadotropic)cellular destruction was observed after this treatment.

Treatment 5 was an application of 50 μM of the fusion peptide modifiedGnRH/hecate (SEQ. ID NO. 3). Widespread basophilic (gonadotropic)cellular destruction was observed after the treatment.

Treatment 6 was an initial application of the fusion peptide GnRH/hecate(SEQ. ID NO. 3), followed by a second application of the fusion peptideGnRH/hecate two hours later. After treatment the tissue was virtuallydestroyed, with only stromal cells remaining.

EXAMPLE 7

Two sexually immature female rats from the same litter (age 33 days)were given two intravenous injections of saline control solution 24hours apart. After the rats reached breeding age, they were examined 105days post-inoculation. The external genitalia appeared normal. During afourteen-day monitoring period 107 days to 121 days post-inoculation,each of the control rats completed at least two estrous cycles. The ratswere then sacrificed and necropsied. The reproductive organs appearedhistologically normal.

EXAMPLE 8

Two sexually immature female rats from the same litter as those ofExample 7 (age 33 days) were given two intravenous injections of 500 μgGnRH/hecate fusion peptide in saline 24 hours apart. After the ratsreached breeding age, they were examined 105 days post-inoculation. Theexternal genitalia appeared small. Unlike the control rats, insertion ofa cotton-tipped swab into the vagina was difficult. During afourteen-day monitoring period 107 days to 121 days post-inoculation,neither of the treated rats demonstrated estrous or metestrous. The ratswere then sacrificed and necropsied. The peptide-treated rats hadthinned, inactive uterine and oviductal epithelia. Their ovariescontained no large follicles, and had a high number of atretic follicles(i.e., those that had failed to ovulate).

EXAMPLES 9-14

Eighteen sexually mature, mixed breed, female rats were randomlyassigned to one of six groups containing three rats each. Each group ofrats received a double treatment intravenously, as described below. Twoweeks after the treatment, the rats were sacrificed and necropsied. Thereproductive and endocrine organs were sectioned, weighed, and examinedhistologically.

Treatment 9 was a saline control. The rats in this group exhibitednormal ovarian function (e.g., normal follicles and new corpora lutea).The pituitaries from this group were of normal size. Histology showed anormal number of pituitary basophilic cells.

Treatment 10 was injection with a total of 1.0 mg GnRH/hecate fusionpeptide in saline, divided into two equal 0.5 mg injections administered24 hours apart. The rats in this group showed an arrest of normalovarian follicular development. Few corpora lutea were present, andthose that were present appeared old. Follicles were large, and had notruptured. Uterine morphology was consistent with hormonal inactivity.The pituitaries from this group were slightly smaller than thepituitaries from the saline control group. Histology revealed a 60% to70% reduction in the number of pituitary basophilic cells compared tothe controls.

Treatment 11 was injection of 100 μL of a 1.35 mM solution of GnRH (162μg) in saline, followed 15 minutes later by injection with 100 μL of a1.35 mM solution of hecate (337 μg) in saline. The same two-steptreatment was repeated 24 hours later. The rats in this group showedaltered ovarian histology. Few corpora lutea were present, and thosethat were present appeared old. Follicles were large, and had notruptured. Uterine morphology was consistent with hormonal inactivity.The pituitaries and the pituitary histology were similar to thoseobserved in Treatment 10.

Treatment 12 was injection of 100 μL of a 1.35 mM solution of hecate(337 μg) in saline. The treatment was repeated after 24 hours. The ratsin this group exhibited normal ovarian function (e.g., normal folliclesand new corpora lutea). The pituitaries and the pituitary histology weresimilar to those observed in Treatment 9.

Treatment 13 was injection of 100 μL of a 1.35 mM solution of GnRH (162μg) in saline. The treatment was repeated after 24 hours. The rats inthis group exhibited normal ovarian function (e.g., normal follicles andnew corpora lutea). The pituitaries and the pituitary histology weresimilar to those observed in Treatment 9.

Treatment 14 was identical to Treatment 10, except that the GnRH/hecatefusion peptide was further purified by HPLC. The rats in this groupshowed an arrest of normal ovarian follicular development. Few corporalutea were present, and those that were present appeared old. Follicleswere large, and had not ruptured. Uterine morphology was consistent withhormonal inactivity. The pituitaries and the pituitary histology weresimilar to those observed in Treatment 10.

These experiments demonstrate that GnRH and the lytic peptide may beadministered either separately or as a fusion peptide, although thefusion peptide is preferred as it is expected to be more active at lowerdoses.

Although experiments to determine optimum dosages had not been performedby the time this application is being filed, a person of ordinary skillin the art, who is given the teachings of the present specification, mayreadily ascertain optimum dosages through routine testing.

Although the experiments to date have been performed on female animals,similar results are expected for male animals, because GnRH signalspituitary cells to release gonadotropins in both males and females.

Tissue and cell specificity of cytotoxic conjugates could be furtherenhanced by using various hormones or hormone analogs coupled to a lyticpeptide. Some examples follow. For fertility control, both the pituitaryand the central GnRH neuronal component of the reproductive axis areselectively damaged by GnRH-hecate conjugate. Few cells in the centralnervous system should be damaged by this treatment, because the chickenII GnRH and lamprey III GnRH forms are the primary molecules affectingbrain function in most mammals. Fertility control may also beselectively accomplished by treating animals with a bLH-hecateconjugate; this compound should specifically affect GnRH neuronscontrolling reproduction and the gonads. To target prostatic, breast,ovarian, or endometrial cancer cells, the I-LHRH-III-hecate conjugatecould be used since it binds to receptors on cancer cells, and has nosignificant known action on the brain. (Other lytic peptides may be usedin place of hecate in these conjugates.)

The compositions of the present invention may be administered asdescribed, or as pharmaceutically acceptable salts. The compositions maybe administered intravenously, subcutaneously, intramuscularly, ororally (especially when in D-amino acid form, preferably complexed witha carrier, e.g., vitamin B₁₂).

Applications of the present invention include long-term contraception orsterilization in humans; and long-term contraception or sterilization indomesticated or wild mammals, birds, reptiles, amphibians, bony fish,cartilaginous fish, jawless fish, and invertebrates such as insects ormolluscs. Domesticated mammals in which this invention may be usedinclude, for example, dogs, cats, cattle, horses, pigs, and sheep. Whenused in humans, long-term replacement hormone therapy may be needed toprevent undesirable side effects, such as premature menopause.Administration of gonadotropic hormones in a sterilized individual willtemporarily restore fertility if desired. The sterilization isreversible in this sense.

As one example, this invention may be used in the humane populationcontrol of an unwanted introduced species.

Sterilization of domesticated birds such as chickens and turkeys canincrease their growth rate. Avian GnRH or analogs may be used inpracticing this invention to sterilize birds. There are two forms ofavian GnRH—Chicken I GnRH (SEQ. ID NO. 17) and Chicken II GnRH (SEQ. IDNO. 18). Either form of avian GnRH may be used in this invention. In apreferred embodiment, position 6 of Chicken I GnRH is linked to a lyticpeptide such as hecate to form a fusion peptide. Alternatively, a GnRHagonist or antagonist may be used. A series of agonists and antagonistshas been synthesized by I. Mezo et al., “Synthesis of GnRH analogshaving direct antitumor and low LH-releasing activity,” Biomed.Peptides, Proteins & Nucleic Acids, vol. 2, pp. 33-40 (1996).

When used to treat insects that are pests to crop plants or otherplants, it may be desirable to incorporate genes encoding thepeptide/ligand combination into the plant's genome, under the control ofa promoter that expresses the peptide in tissues of the plant that areattacked by the insect, but not in tissues that are used for food. Forexample, in a potato a promoter could be used that is active in theleaves of the plant, but not in the tuber. Expression in the planttissue could be constitutive, or alternatively could be induced bystimuli that induce the plant's native defense mechanisms, for exampleby placing the peptide gene under the control of native promoters thatare so induced in plants. See, e.g., U.S. patent application Ser. No.08/279,472, filed Jul. 22, 1994.

When used to sterilize aquatic animals such as fish or molluscs, thecompounds of the present invention may be simply administered in thewater, from which they will be taken up by the animals in adult,juvenile, or larval stages. Preferably, the peptides are encapsulated inliposomes, which are fed to the animals as spat, fry, juveniles, oradults; the animals feed on the liposomes, which then release thecompounds into the animal's circulation, causing sterilization.Alternatively, the peptides may be injected into an animal that hasreached sufficient size.

For example, the compounds may be used to sterilize undesirable exoticmolluscs such as the zebra mussel. Sterilization of aquaculture speciesmay also be desirable. For example, sterilization of oysters willprevent the oysters from ripening gonads in the summer (when they wouldotherwise do so), thereby improving their marketability.

EXAMPLES 15-22

Eight sexually mature, Sprague-Dawley female rats were randomly assignedto one of eight treatments. Each group of rats received a singletreatment intravenously, as described below. Rats were sacrificed andnecropsied either 48 or 96 hours after treatment. The ovaries, uterus,pancreas, liver, spleen, lungs, heart, thyroid, and adrenal glands werefixed in 10% buffered formalin; sectioned; and stained with H&E(hematoxylin and eosin) stain; except that the pituitary glands werestained with PAS (periodic acid-Schiff) stain with no counter-stain. Thetreatments were selected so that each animal received an equimolaramount of the compound with which it was treated.

Treatments 15 and 16 were IV-injection with 674 μg of D-hecate in 200 μLsaline (1.35 mM). The rat in treatment 15 was sacrificed 48 hours afterinjection, and the rat in treatment 16 was sacrificed 96 hours afterinjection. No gross lesions were noted in the organs of either animal.The pituitary glands of both rats contained a normal number ofPAS-positive cells.

Treatments 17 and 18 were IV-injection with 334 μg of GnRH in 200 μLsaline (1.35 mM). The rat in treatment 17 was sacrificed 48 hours afterinjection, and the rat in treatment 18 was sacrificed 96 hours afterinjection. No gross lesions were noted in the organs of either animal.The pituitary glands of both rats contained a normal number ofPAS-positive cells.

Treatments 19-22 were IV-injection with 1 mg GnRH-hecate fusion peptide(SEQ. ID NO. 3) in 100 μL saline (2.7 mM). The rats in treatments 19 and20 were sacrificed 48 hours after injection, and the rats in treatments21 and 22 were sacrificed 96 hours after injection. No gross lesionswere noted in the organs of any of the four animals, other than thepituitary. The pituitary glands of the animals from treatments 19 and 20were slightly enlarged, hyperemic, and edematous. The pituitary glandsof the animals from treatments 21 and 22 were slightly hyperemic, but ofexpected size. The pituitary glands of all four rats showed a markeddepletion of PAS-positive cells; it was estimated that the depletion was80 to 90% compared to those of control groups. (PAS stain preferentiallystains glycopeptides. LH, FSH, TSH, and MSH are glycopeptide hormones;cells containing these hormones stored in their secretory vacuoles stainpositive with PAS.)

It was thus seen that the GnRH-lytic peptide combination causedmorphological and functional alterations in the adult female ratreproductive system, and in preventing sexual maturity in pre-pubertalfemale rats, but that the fusion peptide selectively eliminated aspecific population of PAS-positive staining cells in the pituitary.

EXAMPLE 23

Hecate is an amphipathic lytic peptide that acts on cell membraneswithout being internalized. It is a synthetic peptide analog ofmelittin, the primary toxin in honeybee venom. Hecate is believed to actby disrupting cell membranes. The structure of the modified GnRH-hecateconjugate used in these studies was SEQ. ID NO. 3.

We also synthesized D-Lys⁶GnRH (SEQ. ID NO. 13), so that hecate could beconjugated to the D-Lys⁶, a position that could minimize interferencewith binding of the GnRH domain to the GnRH receptor. These syntheticpeptides specifically displaced radiolabelled monoiodinated-GnRH fromrat pituitary membranes. Displacement by D-Lys⁶GnRH-hecate wascomparable to (and actually slightly greater than) displacement bynative mammalian GnRH, as measured by cpm of radioactivity. When GnRHand GnRH-hecate binding were compared on a molar basis over a 1000-foldconcentration range (n=6) the GnRH-hecate specifically displaced theradiolabelled peptide to an extent equal to 123%±4% of the bindingexhibited by equimolar concentrations of GnRH; equimolar concentrationsof D-Lys⁶GnRH displaced 187%±8% of the cpm displaced by native GnRH.

EXAMPLES 24-31

We studied in vitro lysis of bovine luteal cells with GnRH-hecateconjugate and with hecate-bLH conjugate (SEQ. ID NO. 12). (The bLHcomponent of the conjugate is a 15-mer fragment of the beta chain ofluteinizing hormone, SEQ. ID NO. 11). Small luteal cells were collectedfrom cattle corpora lutea post-slaughter. Small luteal cells are rich inLH receptors, and were found to be highly susceptible to lysis by thehecate-bLH conjugate.

Small luteal cells in culture were incubated with one of the followingtreatments for 22 hours, and were then examined for viability usingTrypan Blue exclusion and release of lactic dehydrogenase.

Treatment 24 control: no additional treatment (media alone)

Treatment 25 10 ng bLH (positive control)

Treatment 26 hecate-bLH, 10 μM

Treatment 27 hecate-bLH, 5 μM

Treatment 28 hecate-bLH, 1 μM

Treatment 29 hecate (alone), 10 μM

Treatment 30 hecate (alone), 5 μM

Treatment 31 hecate (alone), 1 μM

Significant killing of small luteal cells was observed following 22 hr.incubation with 10 μM hecate alone, and with 5 μM hecate alone(approximately 50% killing). Cell death for 1 μM hecate alone did notdiffer significantly from negative control (media) or from bLH alone.All three treatment doses with hecate-bLH caused significant increasesin cell death as compared to treatment with hecate alone. The hecate-bLHconjugate killed approximately twice the number of cells as were killedby hecate alone at the same concentrations.

Observed levels of lactic dehydrogenase activity also demonstrated thatthe hecate-bLH treatment killed a significantly greater number of cellsthan did hecate alone.

EXAMPLES 32-33

We also studied in vitro lysis of bovine granulosa cells withGnRH-hecate conjugate and with hecate-bLH conjugate. Granulosa cellswere isolated from bovine pre-ovulatory follicles. (Granulosa cells arehormonally active cells with numerous LH receptors.) Our experimentswith granulosa cells were otherwise generally similar to those describedabove for Examples 24-31. These experiments demonstrated (1) that thegranulosa cells were much more susceptible to killing by hecate alonethan were the small luteal cells, and (2) that, as had been the casewith the small luteal cells, the granulosa cells were significantly moresusceptible to hecate-bLH at even the lowest concentration (1 μM) thanthey were to hecate alone. At 1 μM, the hecate-bLH conjugate killedabout twice the number of target cells as did hecate alone. Again, thelevels of lactic dehydrogenase released following the hecate-bLH 1 μMtreatment were significantly higher than the levels of enzyme releasedfollowing treatment with 1 μM hecate alone.

Additional studies (data not shown) demonstrated that a 15-mer fragmentof the bLH subunit specifically bound to LH receptors on the targetgranulosa cells, but did not initiate the production of steroid hormonesthat would be indicative of a stimulus-coupled response. We thusdemonstrated that the selective killing of target cells resulted fromthe physical proximity of the lytic peptide to the cell, which wascaused by binding of the LH subunit. Stimulation of target cell hormoneproduction was not required for cell lysis. This result was surprising,as we had previously expected that activation of the target cells wasrequired for increased susceptibility to lysis. These data demonstratethat such activation is not required. These data are, however,consistent with our other data showing that cell activation is also aroute that can lead to increased susceptibility to the lytic peptide.

EXAMPLES 34-37

Another set of experiments was performed to study the in vivo effects ofthe GnRH-hecate conjugate on female rats and rabbits. The ovaries,uterus, oviducts, adrenals, spleen, thyroids, pancreas, liver, lungs,and heart were processed for histological analysis. The pituitaries wereprocessed for histological analysis of PAS-stained cells and for cellsstained immunocytochemically for bLH, BFSH (bovine follicle stimulatinghormone), adrenocorticotropic hormone, and other proopiomelanocortinpeptide products (most notably alpha-melanocyte stimulating hormone(MSH)), thyroid stimulating hormone (TSH), prolactin (PRL), vasopressin(VP), oxytocin (OXY) or growth hormone (GH). The immunocytochemicalstaining procedures we used followed generally the procedures of M.Rahmanian et al., “Histological and immunocytochemical characterizationof pituitary cell types in ponies,” Proc. 13th Soc. Equine Nutrition &Phys. Symp., pp. 348-349 (1993); M. Rahmanian et al.,“Immunocytochemical localization of luteinizing hormone andfollicle-stimulating hormone in the equine pituitary,” J. Anim. Sci.,vol. 76, pp. 839-846 (1998); M. Rahmanian et al., “Immunocytochemicallocalization of prolactin and growth hormone in the equine pituitary,”Animal Sci., vol. 75, pp. 3010-3018 (1997); and P. Melrose et al.,“Comparative topography of the immunoreactivealpha-melanocyte-stimulating hormone neuronal system in the brains ofhorses and rats,” Brain Beh. & Evol., vol. 32, pp. 226-235 (1988).

Brains were serially sectioned on a Vibrotome from the level of thediagonal band of Broca to the mammillary body. Alternate sections wereconsecutively divided into four to five dishes, and sections inalternate dishes were stained with cresyl violet, or were stainedimmunocytochemically for GnRH or the GnRH precursor, VP, OXY, ortyrosine hydroxylase (the rate-limiting enzyme in catecholaminesynthesis). In addition to the staining procedures cited above, we alsoused the immunocytochemical staining procedures of P. Melrose et al.,“Distribution and morphology of immunoreactive gonadotropin-releasinghormone (GnRH) neurons in the basal forebrain of ponies,” J. Comp.Neurol. vol. 339, pp. 269-287 (1994); and P. Melrose et al., “Topographyof oxytocin and vasopressin neurons in the forebrain of Equus caballus:Further support of proposed evolutionary relationships forproopiomelanocortin, oxytocin and vasopressin neurons,” Brain, Beh. &Evol., vol. 33, pp. 193-204 (1989).

Thirty-three-day-old, sexually immature female rats were givenintravenous administrations as follow:

Treatment 34: 0.03 μg GnRH (a normal physiological dose) (two rats)

Treatment 35: 1.62 μg GnRH (the molar equivalent to the amount of GnRHin Treatment 36) (one rat)

Treatment 36: 0.5 mg GnRH-hecate (one rat)

Treatment 37: 0.03 μg GnRH, followed 11 minutes later by 0.337 μg hecate(two rats).

Animals were sacrificed 14 days after treatment. As compared to the twoGnRH control groups, the treatment with GnRH-hecate and the treatmentwith GnRH followed by hecate alone reduced pituitary weights by 13% and14%, respectively, and reduced the numbers of bLH-specific gonadotropesby 92% and 87%, respectively. Further, following these two experimentaltreatments the cell bodies of GnRH-stained neurons in hypophysiotropicareas of the brain were frequently deformed; and a substantial amount ofimmunoreactive material leached into surrounding areas where numerouscell bodies are concentrated (the organum vasculosum of the laminaterminalis). There was histological damage to cells from the twoexperimental treatments in the C1 and C3 fields of the hippocampus, andincreased staining of parvicellular VP neurons in the paraventricularnucleus. (The VP staining may have been caused by formation of aprecipitate in certain areas of the brain. Subsequent studies with morehighly purified peptide did not show a precipitate). The change in VPexpression, probably in corticotropin-releasing neurons, may cause ashift in the post-translational processing of proopiomelanocortinpeptide products in the pars distalis, since GnRH-hecate and GnRH+hecatetreatments reduced adrenocorticotropic hormone levels and increased thenumber of alpha-MSH-stained cells in this subdivision of the pituitary.No pathological changes were noted in any other tissues.

Since neurons in the brain do not regenerate, severe damage to theseneurons could make sterilization with a GnRH/lytic peptide combinationpermanent (but temporarily reversible by administration of gonadotrophichormones).

EXAMPLES 38-42

Sexually immature (33 day old) female rats (randomly allocated intogroups of three) were injected intravenously with saline or GnRH-hecatein saline as follows:

Treatment 38: 0.0 mg GnRH-hecate

Treatment 39: 0.1 mg GnRH-hecate

Treatment 40: 0.5 mg GnRH-hecate

Treatment 41: 1.0 mg GnRH-hecate

Treatment 42: 1.5 mg GnRH-hecate.

Animals were sacrificed at 24 hours or at 14 days after treatment.Results were similar to those reported above for Examples 34-37, exceptthat no precipitate was found in the brain, and VP staining in the CNSwas not altered. The treatments with higher levels of GnRH-hecateproduced a large number of GnRH-receptor-containing neurons havingabnormal morphologies, including distortion of the somatic portion ofthe cells, and degeneration of neurites. In the rats sacrificed fourteendays after treatment, 66% and 87% of the GnRH-receptor-containingneurons were abnormal in the rats that had received 1.0 and 1.5 mg ofGnRH-hecate, respectively. Axonal degeneration in the 1.5 mg GnRH-hecategroup was accompanied by over 90% reduction in median eminence stainingfor GnRH.

EXAMPLES 43-45

Seven sexually mature female New Zealand rabbits were injectedintravenously with saline containing GnRH-hecate as follows:

Treatment 43: 0 mg GnRH-hecate (n=3)

Treatment 44: 5 mg GnRH-hecate (n=3)

Treatment 45: 10 mg GnRH-hecate (n=1).

Forty-six days later all rabbits were injected intramuscularly with 100μg GnRH. Blood samples were collected at 0, 1, 4, and 24 hours, and LHand FSH levels in the blood samples were measured by radioimmunoassay.Hormone analyses revealed that both control and experimental animalsreleased LH in response to the GnRH, suggesting that there may be atleast some degree of reversibility following treatment, at least forpituitary gonadotropes at lower doses of ligand/peptide. The rabbitswere sacrificed the next day (day 47) for postmortem histologicalanalysis. We found that the numbers of tertiary follicles, corporalutea, and GnRH-induced ovulations were reduced by GnRH-hecatetreatment. Ovarian and pituitary weights were reduced by the 10 mgGnRH-hecate treatment. In tissues from the GnRH-hecate treatments,observed immunoreactive GnRH was faint and diffusely localized in CNSareas normally containing cell bodies; normal individual cell bodieswere reduced in number by at least 50%; and the terminal fields, whichnormally contain the axons of GnRH receptor neurons, were not stainedfor GnRH. These observations suggest that the most pronounced effects ofthe GnRH-hecate treatments in these experiments on rabbits may have beenon neuroendocrine neurons in the brain. The hippocampus and other areasof the brain containing high concentrations of GnRH were not discerniblyaffected by GnRH-hecate treatments. The GnRH-hecate treatment increasedthe number of PAS-stained pituitary cells in the pars distalis to 177%of that for control rabbits; this increase appeared to reflect increasednumbers of cells staining alpha-MSH, and reduced numbers of cellsstaining for LH.

EXAMPLES 46-47

Nine sexually mature female rabbits were injected intravenously withsaline containing 0 mg (n=4) (Treatment 46) or 10 mg GnRH-hecate (n=5)(Treatment 47). Rabbits were injected intramuscularly with GnRH on day 6posttreatment. Blood samples were collected for radioimmunoassay of LHand FSH as described above, and the animals were sacrificed on day 7post-treatment. Both control and experimental animals released LH inresponse to the GnRH; however, the amount of LH released was lower inthe treated animals than in the controls. The GnRH-hecate treatmentreduced the numbers of tertiary ovarian follicles, and the numbers ofGnRH-induced ovulations. No effects were noticed either on peripheraltissues or on pituitary weight. The effects of GnRH-hecate on CNSmorphology and immunocytochemical results were similar to thosedescribed above in Examples 34-45. Again, the effects were morepronounced on GnRH neurons than on staining of pituitary gonadotropes.

The number of ovulation sites in rabbits in Examples 46 and 47 treatedwith 10 mg GnRH-hecate were reduced as compared to saline controls. Themean number of ovulation sites in four saline controls equalled12.2±5.4, with S.E.M.=2.7. The mean number of ovulation sites in thefive rabbits given 10 mg of GnRH-hecate was 3.6±1.1, with S.E.M.=0.5.This difference from control was significant (p=0.025).

The “LH surge” (the level of LH at one hour post-GnRH challenge, minusthe resting level before challenge) in the four controls was 61.2±16.5ng/mL, with S.E.M.=8.3; and in the treated group was 49.6±26.1 ng/mL,with S.E.M.=12 (p=0.22). Thus there was a trend towards lower LH levelsin the treated group.

The in vivo studies clearly demonstrated that the GnRH-hecate conjugateselectively damaged GnRH receptor-bearing cells in the brain (neurons)and in the pituitary (gonadotrophic cells). Further, these studiesdemonstrated a significant alteration in the ovary, presumably aconsequence of alteration in the reproductive centers of thebrain-pituitary axis. Selectivity of the conjugate was demonstrated bythe following observations: (1) No cytotoxic changes were seen inneurons that lacked GnRH receptors. (2) No changes were seen inpituitary cells that lacked GnRH receptors. (3) No changes were seen inother endocrine and non-endocrine tissues (except for the ovary, whichpresumably responded indirectly to the destruction of gonadotrophs inthe pituitary).

Many of the events referred to as “ovulations” in the GnRH-hecatetreated rabbits possibly were not functional ovulation sites, but mayinstead have represented hemorrhagic pre-ovulatory degenerative changes.Additional breeding trials will be conducted to verify that ovulation offunctional ova is prevented.

EXAMPLES 48-51

The following examples demonstrated the ability of a GnRH-lytic peptidecombination to reduce fertility in insects. It was also unexpectedlydiscovered that the lytic peptide alone (i.e., administered withoutGnRH) had similar effects. Although insects are not believed to secretea GnRH identical to that found in mammals, there appears to be somehomology, in that the insects did respond to mammalian GnRH, and to GnRHlinked to the lytic peptide hecate.

Late-stage Diatraea saccharalis (sugar cane borer) pupae were inoculatedwith 1.0 μL of saline solution containing 1.35 mM concentration ofpeptide as stated, or saline alone as control. The pupae were allowed tocomplete metamorphosis. No gross morphological defects were observed inany of the insects completing metamorphosis. Adult female moths wereallowed to mate with treated males, and then lay eggs. The viability ofthe eggs was measured by counting the number hatching into larvae.

Treatment 48 was the control, inoculation of 21 pupae with saline alone.Twelve of the pupae completed metamorphosis into adult moths (4 males, 8females). The 8 females laid a total of about 900 eggs, an average ofabout 112 eggs per female. About 22% of these eggs hatched, or about 25hatched larvae per female.

Treatment 49 was inoculation of 10 pupae with 1.35 mM GnRH. Four of thepupae completed metamorphosis into adult moths (2 males, 2 females). The2 females laid a total of about 300 eggs, or an average of about 150eggs per female. About 40% of these eggs hatched, or about 60 hatchedlarvae per female.

Treatment 50 was inoculation of 10 pupae with 1.35 mM GnRH-hecate (SEQ.ID NO. 3). Eight of the pupae completed metamorphosis into adult moths(3 males, 5 females). The 5 females laid a total of about 200 eggs, oran average of about 40 eggs per female. About 40% of these eggs hatched,or about 16 hatched larvae per female.

Treatment 51 was inoculation of 10 pupae with D-hecate. Six of the pupaecompleted metamorphosis into adult moths (2 males, 4 females). The 4females laid a total of 18 eggs, or an average of 4.5 eggs per female.100% of these eggs hatched, or 4.5 hatched larvae per female.

It was thus observed that, compared to controls, females treated withGnRH alone in the late pupal stage had enhanced reproductive success;those treated with the GnRH-hecate combination had decreasedreproductive success; and those treated with D-hecate alone had evenlower reproductive success.

Without wishing to be bound by the following hypothesis, it is believedthat these results may be explained as follows. Due to (as yetunidentified) sequence homology across taxa, small peptides active inthe control of mammalian reproduction also influence reproductivefunction in insects. See W. Theunis et al., “Luteinising Hormone,Follicle Stimulating Hormone and Gonadotropin Releasing HormoneImmunoreactivity in Two Insects: Locusta migratoria migratoroides R & Fand Sarcophaga bullata (Parker),” Invert. Reprod. and Develop., vol. 16,pp. 111-117 (1989); and P. Verhaert et al., “Substances ResemblingPeptides of the Vertebrate Gonadotropin System Occur in the CentralNervous System of Periplaneta americana L.,” Insect Biochem., vol. 16,pp. 191-197 (1986).

This activity is probably mediated by the inherent ability of thesepeptides to react with appropriate intermediate cells by aligand-receptor interaction, thus altering the functional activity ofthe intermediate cells. More particularly, insects have a receptor thatresponds to mammalian GnRH. GnRH alone stimulates reproductive activityin insects. GnRH coupled to a lytic peptide attacks the intermediatecells in the insects, inhibiting reproductive activity.

The results observed for the D-hecate administered without GnRH weresurprising, and are explained somewhat differently, again withoutwishing to be bound by the following hypothesis. Metamorphosis is a timeof high cell activity. Lytic peptides generally have greater activityagainst active cells. The observed response to hecate alone is believedto be a generalized response by activated cells, not a specific responsemediated by a receptor. The fact that the D-conformation of hecate wasused in this experiment may be significant, since D-form peptidesgenerally have a longer biological half-life. It is currently unknownwhether similar results would be seen with L-hecate alone. (D-hecate wasused in Treatment 26 for the simple reason that previously-synthesizedD-hecate was readily available to the investigators.)

Treatment of Malignant and Benign Tumors

The compositions of the present invention are useful in killing orinhibiting the growth of malignant and benign tumor cells that expressreceptors for GnRH, LH, hCG, I-LHRH-III, or steroids. The ligand isadministered with a lytic peptide (either sequentially, or linked to oneanother), and the targeted tumor cells are killed or inhibited.

In treating hormone- or ligand-linked cancers (e.g., cancers of theovary, testis, breast, uterus, endometrium, pituitary, and prostate),lytic peptides may be attached to the hormone for which the tumorexpresses a receptor or set of receptors, e.g., an estrogen,testosterone, LH, FSH, estradiol-17β, transforming growth factor alpha(TGFα), epidermal growth factor (EGF), GnRH, LH, hCG, lamprey III LHRH(I-LHRH-III), and melanocyte stimulating hormone. For example, an esterlinkage of a lytic peptide to estradiol or testosterone can convenientlybe made by condensing the carboxy terminus of the lytic peptide with thehydroxyl group at the 17-carbon position of the steroid. Anestradiol/lytic peptide combination may be used as a treatment againstbreast or ovarian cancer; and a testosterone/lytic peptide combinationmay be used to treat prostate cancer. In addition, the specific bindingdomains of the peptide hormone LH or FSH may be used in fusion peptideswith a lytic peptide to selectively bind the fusion peptide to targettumor cells with cell surface receptors for these hormones. For example,the receptor binding site of the β-subunit of LH and hCG may be used(SEQ. ID NO. 11). See Morbeck et al., Mol. and Cell Endocrin., vol. 97,pp. 173-186 (1993).

Pituitary Tumors

The anterior pituitary contains different types of epithelial cells thatcontrol the complex processes of growth, reproduction, lactation,thyroid function, and adrenal functions. Due to the high functionalplasticity of pituitary cells (i.e., their ability to differentiate intodifferent cellular phenotypes in response to stimuli), these cells areparticularly prone to aberrant behavior. Because many of the signals towhich the pituitary responds are receptor-mediated, pathological statesmay be controlled by co-opting the appropriate ligand-receptorinteraction. Several examples are given below.

Dopamine Receptors in Prolactinomas and Other Adenomas

Chronic dopamine deficiency has been associated with some types ofpituitary tumors. In certain adenomas the number of dopamine bindingsites is reduced by about 50%, and the number can be reduced evenfurther during dopaminergic therapy. It has also been reported thatnerve growth factor can stimulate prolactinoma cells to re-expressdopamine receptors. Pretreating a prolactinoma with nerve growth factorbefore treatment with a dopamine/lytic peptide combination makes itsusceptible to treatment through the present invention. The lyticpeptide may be linked to dopamine, for examine, by an amide group formedby condensing the carboxy terminus of the peptide with the amino groupof dopamine.

This therapy will be effective not only for prolactinomas, but also forother adenomas expressing dopamine receptors, such as growthhormone-secreting adenomas, thyrotropin-releasing hormone secretingadenomas, and gonadotropin-secreting adenomas.

Somatostatin Receptors in Growth Hormone-Secreting Adenomas

It has been reported that growth hormone (GH)-secreting adenomas have ahighly variable number of somatostatin receptors. (Variation by at leasta factor of 10 may be seen among individual tumors.) There is alsoconsiderable variation in the distribution of binding sites: thesomatostatin receptors may be homogeneously distributed, locatedexclusively in one portion of the tumor tissue, or in between.

Somatostatin receptors are also present in other types of pituitarytumors. It has been reported that the cell surfaces of a majority of GH-and thyrotropin releasing hormone (TRH)-secreting adenomas have anelevated number of somatostatin receptors.

Such tumors may be treated by the present invention by asomatostatin/lytic peptide combination.

Other Pituitary Adenomas

Other ligands that may be used in a ligand/lytic peptide combination totreat other pituitary adenomas include TRH, MSH, GnRH,corticotropin-releasing hormone, growth hormone-releasing hormone,vasoactive intestinal polypeptide, and pituitary adenylate cyclaseactivating peptide. A short chain analog of αMSH that may be used inplace of MSH is Ser-Tyr-Cys-Met-Glu-His-Phe-Arg-Trp-Asn-Lys-Pro-Val-NH₂(SEQ. ID NO. 10).

Other Endocrine-Related Diseases

In other applications, the ligand/lytic peptide combination of thepresent invention may be used to treat endocrine-related diseasesgenerally. Where a disease is causally related to dysfunction of cellshaving certain hormone receptors, cells with such receptors may beselectively inactivated by administering a combination of the hormoneand a lytic peptide.

In an alternative approach, it has previously been noted that it isbeneficial to reduce levels of LH and FSH in breast and prostate cancerpatients. If the gonadotropes in the pituitary are selectively killedwith a GnRH/lytic peptide combination, then the pituitary will no longersecrete LH and FSH. The reduced levels of these hormones thus resultingwill help control the spread of the cancers. This alternative, indirectapproach may be used in lieu of, or in addition to, treating the cancersdirectly with a LH/lytic peptide or FSH/lytic peptide combination.Chronic administration of GnRH has previously been used to down-regulateits receptors, and thus effectively remove LH from circulation,resulting in “chemical castration” of prostatic cancer patients.However, GnRH and certain GnRH analogs also have direct effects onprostatic cell growth.

By analogy, it is well-established that surgical removal of the anteriorpituitary is effective in treating sex hormone-related diseases.Chemical destruction of gonadotrophic cells in the pituitary through thepresent invention will therefore have similar effects on sexhormone-related diseases, but without the attendant risks andcomplications of surgery.

EXAMPLES 52-58

In these experiments we demonstrated in vitro lysis of human prostatecancer cell lines. LNCaP FGC and DU145 human prostate cancer cell lineswere purchased from the American Type Culture Collection (ATCC,Rockville Md.), ATCC accession numbers CRL 1740 and HTB-81,respectively. The LNCaP FGC adenocarcinoma cell line was originallyobtained from a 50 year old male Caucasian. LNCaP FGC cells aresensitive to dihydrotestosterone and to estrogens (A+). The DU145carcinoma was originally isolated from the brain of a 69 year old maleCaucasian with metastatic carcinoma of the prostate; this cell line isnot sensitive to steroid hormones (A−).

Cells were detached from culture flasks, and 1000 cells/well weretransferred to 24 well culture plates. The cells were incubated for 24hours with 10% calf serum. Cells were subsequently incubated withoutserum for 48 hours. Cells were then incubated for 22 hours with one ofthe following treatments:

Treatment 52: 10 μM luteinizing hormone (LH)

Treatment 53: 30 μM free hecate

Treatment 54: 90 μM hecate-bLH

Treatment 55: 60 μM hecate-bLH

Treatment 56: 50 μM GnRH-hecate

Treatment 57: 10 μM GnRH-hecate

Treatment 58: FSH pre-treatment, followed by 90 μM hecate-bLH

Trypan blue exclusion was used to assess viability of the cells aftertreatment. The treatment that most consistently and effectively killedboth the A+ and the A− cancer cell lines was the higher dose (50 μM) ofGnRH-hecate. The lower dose (10 μM) of GnRH-hecate was equally effectiveagainst the androgen-insensitive DU145 cells. The DU145 cells were alsokilled by hecate alone. However, treatment with a lytic peptide alonemay not be selective in vivo unless specific cell types are separatelystimulated, for example by hormones controlling their activity. Thehecate-bLH conjugate killed almost all DU145 cells, but had littleeffect on A+ LNCaP. This result is consistent with specific binding ofLH to DU145 cells but not to LNCaP cells. LH specifically binds DU145cells, but we have not been able to consistently measure specificbinding of LH to the A+ LNCaP cells. The LNCaP cells pre-treated withFSH were more sensitive to the hecate-bLH conjugate than those that werenot pre-treated.

Other Applications, including Treatment of Autoimmune Diseases, andTargeting of Abnormal Cells

This invention may be used wherever it is desirable to specificallyinhibit abnormal (or normal) cells that are driven by or are dependenton specific ligand interactions. As another example, this invention maybe used in treating autoimmune diseases for which the antigen or epitoperesponsible for the autoimmune disease is known.

Specific immune responses are mediated by B-lymphocytes, T-lymphocytes,or both. When lymphocytes inappropriately attack “self” instead of“non-self,” a variety of autoimmune diseases can result, some of whichcan have devastating consequences. Diseases that have been associatedwith autoimmunity include rheumatoid arthritis, juvenile rheumatoidarthritis, systemic lupus erythematosus, Addison's disease,Goodpasture's syndrome, autoimmune hemolytic anemia, Grave's disease,Hashimoto's thyroiditis, idiopathic thrombocytopenia purpura,insulin-dependent diabetes mellitus, myasthenia gravis, myocardialinfarction, aplastic anemia, pernicious anemia, poststreptococcalglomerulonephritis, spontaneous infertility, ankylosing spondylitis,scleroderma, and Sjögrens' syndrome.

Whether mediated by T-cells or B-cells, autoimmune disease ischaracterized by lymphocytes with specific receptors for a self epitopethat triggers their function—i.e., antibody secretion, proliferation,secretion of cytotoxic factors, or secretion of inflammatory cytokines.These responses cause damage or destruction to self cells or organs.

The specific antigens and even epitopes that act as ligands to stimulatethe lymphocytes have been identified for several autoimmune diseases,typically by the in vitro proliferative response they induce inlymphocytes. For example, thyrotropin has been implicated as theself-antigen recognized by lymphocytes in Hashimoto's Disease. Where theepitope is known, the autoimmune disease may be treated by administeringa compound containing that epitope linked to a lytic peptide, which willselectively delete clones of the autoreactive lymphocytes.

There have previously been no general treatments for autoimmunediseases. Prior treatments have included cytotoxic compounds, and highdoses of corticosteroids, both of which have risks in long-term therapy.Neither selectively targets autoreactive lymphocytes.

Certain abnormal cells (e.g., virally-infected cells such asHIV-infected cells, cancer cells) display surface receptors that are notfound on normal cells. In some cases, these receptors are encoded byviral nucleic acids. Ligands for these receptors, such as monoclonalantibodies to those receptors, or the receptor/ligand pairs shown inTable 2 of D. Fitzgerald et al., “Targeted Toxin Therapy for theTreatment of Cancer,” J. Natl. Cancer Inst., vol. 81, pp. 1455-1463, maybe used in the ligand/lytic peptide combination of the present inventionto selectively destroy cells displaying the receptor. Destruction ofsuch a virally-infected cell, for example, before completion of theviral maturation cycle results in the release of incomplete,non-infectious viral particles, thereby treating the viral infection.Destruction of such a cancer cell prevents further metastasis. Where anantibody is used as the ligand, it will often be preferable toadminister the antibody and the lytic peptide sequentially, rather thanlinked to one another. Complement and other responses to the boundantibodies make the cells more susceptible to attack by the lyticpeptides.

Lytic Peptides Useful in the Present Invention

It is believed (without wishing to be bound by this theory) that lyticpeptides act by disrupting cell membranes. “Resting” eukaryotic cellsprotect themselves through their ability to repair the resultingmembrane damage. By contrast, activated cells (e.g., cells stimulated byGnRH) are unable (or less able) to repair damaged membranes. BecauseGnRH-activated pituitary cells have a diminished capacity to repairmembranes, they are preferentially destroyed by lytic peptides, whileadjacent non-activated cells repair their membranes and survive.

Although the embodiments of this invention that have been tested to datehave used hecate as the effector lytic peptide, this invention will workwith a combination of a ligand with other lytic peptides as well. Manylytic peptides are known in the art and include, for example, thosementioned in the references cited in the following discussion.

Lytic peptides are small, basic peptides. Native lytic peptides appearto be major components of the antimicrobial defense systems of a numberof animal species, including those of insects, amphibians, and mammals.They typically comprise 23-39 amino acids, although they can be smaller.They have the potential for forming amphipathic alpha-helices. See Bomanet al., “Humoral immunity in Cecropia pupae,” Curr. Top. Microbiol.Immunol. vol. 94/95, pp. 75-91 (1981); Boman et al., “Cell-free immunityin insects,” Annu. Rev. Microbiol., vol. 41, pp. 103-126 (1987);Zasloff, “Magainins, a class of antimicrobial peptides from Xenopusskin: isolation, characterization of two active forms, and partial DNAsequence of a precursor,” Proc. Natl. Acad. Sci. USA, vol. 84, pp.3628-3632 (1987); Ganz et al., “Defensins natural peptide antibiotics ofhuman neutrophils,” J. Clin. Invest., vol. 76, pp. 1427-1435 (1985); andLee et al., “Antibacterial peptides from pig intestine: isolation of amammalian cecropin,” Proc. Natl. Acad. Sci. USA, vol. 86, pp. 9159-9162(1989).

Known amino acid sequences for lytic peptides may be modified to createnew peptides that would also be expected to have lytic activity bysubstitutions of amino acid residues that preserve the amphipathicnature of the peptides (e.g., replacing a polar residue with anotherpolar residue, or a non-polar residue with another non-polar residue,etc.); by substitutions that preserve the charge distribution (e.g.,replacing an acidic residue with another acidic residue, or a basicresidue with another basic residue, etc.); or by lengthening orshortening the amino acid sequence while preserving its amphipathiccharacter or its charge distribution. Lytic peptides and their sequencesare disclosed in Yamada et al., “Production of recombinant sarcotoxin IAin Bombyx mori cells,” Biochem. J., vol. 272, pp. 633-666 (1990); Taniaiet al., “Isolation and nucleotide sequence of cecropin B cDNA clonesfrom the silkworm, Bombyx mori,” Biochimica Et Biophysica Acta, vol.1132, pp. 203-206 (1992); Boman et al., “Antibacterial and antimalarialproperties of peptides that are cecropin-melittin hybrids,” FebsLetters, vol. 259, pp. 103-106 (1989); Tessier et al., “Enhancedsecretion from insect cells of a foreign protein fused to the honeybeemelittin signal peptide,” Gene, vol. 98, pp. 177-183 (1991); Blondelleet al., “Hemolytic and antimicrobial activities of the twenty-fourindividual omission analogs of melittin,” Biochemistry, vol. 30, pp.4671-4678 (1991); Andreu et al., “Shortened cecropin A-melittin hybrids.Significant size reduction retains potent antibiotic activity,” FebsLetters, vol. 296, pp. 190-194 (1992); Macias et al., “Bactericidalactivity of magainin 2: use of lipopolysaccharide mutants,” Can. J.Microbiol., vol. 36, pp. 582-584 (1990); Rana et al., “Interactionsbetween magainin-2 and Salmonella typhimurium outer membranes: effect ofLipopolysaccharide structure,” Biochemistry, vol. 30, pp. 5858-5866(1991); Diamond et al., “Airway epithelial cells are the site ofexpression of a mammalian antimicrobial peptide gene,” Proc. Natl. Acad.Sci. USA, vol. 90, pp. 4596 ff (1993); Selsted et al., “Purification,primary structures and antibacterial activities of β-defensins, a newfamily of antimicrobial peptides from bovine neutrophils,” J. Biol.Chem., vol. 268, pp. 6641 ff(1993); Tang et al., “Characterization ofthe disulfide motif in BNBD-12, an antimicrobial β-defensin peptide frombovine neutrophils,” J. Biol. Chem., vol. 268, pp. 6649 ff(1993); Lehreret al., Blood, vol. 76, pp. 2169-2181 (1990); Ganz et al., Sem. Resp.Infect. I., pp. 107-117 (1986); Kagan et al., Proc. Natl. Acad. Sci.USA, vol. 87, pp. 210-214 (1990); Wade et al., Proc. Natl. Acad. Sci.USA, vol. 87, pp. 4761-4765 (1990); Romeo et al., J. Biol. Chem., vol.263, pp. 9573-9575 (1988); Jaynes et al., “Therapeutic AntimicrobialPolypeptides, Their Use and Methods for Preparation,” WO 89/00199(1989); Jaynes, “Lytic Peptides, Use for Growth, Infection and Cancer,”WO 90/12866 (1990); Berkowitz, “Prophylaxis and Treatment of AdverseOral Conditions with Biologically Active Peptides,” WO 93/01723 (1993).

Families of naturally-occurring lytic peptides include the cecropins,the defensins, the sarcotoxins, the melittins, and the magainins. Bomanand coworkers in Sweden performed the original work on the humoraldefense system of Hyalophora cecropia, the giant silk moth, to protectitself from bacterial infection. See Hultmark et al., “Insect immunity.Purification of three inducible bactericidal proteins from hemolymph ofimmunized pupae of Hyalophora cecropia,” Eur. J. Biochem., vol. 106, pp.7-16 (1980); and Hultmark et al., “Insect immunity. Isolation andstructure of cecropin D. and four minor antibacterial components fromcecropia pupae,” Eur. J. Biochem., vol. 127, pp. 207-217 (1982).

Infection in H. cecropia induces the synthesis of specialized proteinscapable of disrupting bacterial cell membranes, resulting in lysis andcell death. Among these specialized proteins are those knowncollectively as cecropins. The principal cecropins—cecropin A, cecropinB, and cecropin D—are small, highly homologous, basic peptides. Incollaboration with Merrifield, Boman's group showed that theamino-terminal half of the various cecropins contains a sequence thatwill form an amphipathic alpha-helix. Andrequ et al., “N-terminalanalogues of cecropin A: synthesis, antibacterial activity, andconformational properties,” Biochem., vol. 24, pp. 1683-1688 (1985). Thecarboxy-terminal half of the peptide comprises a hydrophobic tail. Seealso Boman et al., “Cell-free immunity in Cecropia,” Eur. J. Biochem.,vol. 201, pp. 23-31 (1991).

A cecropin-like peptide has been isolated from porcine intestine. Lee etal., “Antibacterial peptides from pig intestine: isolation of amammalian cecropin,” Proc. Natl. Acad. Sci. USA, vol. 86, pp. 9159-9162(1989).

Cecropin peptides have been observed to kill a number of animalpathogens other than bacteria. See Jaynes et al., “In Vitro CytocidalEffect of Novel Lytic Peptides on Plasmodium falciparum and Trypanosomacruzi,” FASEB, 2878-2883 (1988); Arrowood et al., “Hemolytic propertiesof lytic peptides active against the sporozoites of Cryptosporidiumparvum,” J. Protozool., vol. 38, No. 6, pp. 161 S-163S (1991); andArrowood et al., “In vitro activities of lytic peptides against thesporozoites of Cryptosporidium parvum,” Antimicrob. Agents Chemother.,vol. 35, pp. 224-227 (1991). However, normal mammalian cells do notappear to be adversely affected by cecropins, even at highconcentrations. See Jaynes et al., “In vitro effect of lytic peptides onnormal and transformed mammalian cell lines,” Peptide Research, vol. 2,No. 2, pp. 1-5 (1989); and Reed et al., “Enhanced in vitro growth ofmurine fibroblast cells and preimplantation embryos cultured in mediumsupplemented with an amphipathic peptide,” Mol. Reprod. Devel., vol. 31,No. 2, pp. 106-113 (1992).

Defensins, originally found in mammals, are small peptides containingsix to eight cysteine residues. Ganz et al., “Defensins natural peptideantibiotics of human neutrophils,” J. Clin. Invest., vol. 76, pp.1427-1435 (1985). Extracts from normal human neutrophils contain threedefensin peptides: human neutrophil peptides HNP-1, HNP-2, and HNP-3.Defensin peptides have also been described in insects and higher plants.Dimarcq et al., “Insect immunity: expression of the two major inducibleantibacterial peptides, defensin and diptericin, in Phormia terranvae,”EMBO J., vol. 9, pp. 2507-2515 (1990); Fisher et al., Proc. Natl. Acad.Sci. USA, vol. 84, pp. 3628-3632 (1987).

Slightly larger peptides called sarcotoxins have been purified from thefleshfly Sarcophaga peregrina. Okada et al., “Primary structure ofsarcotoxin I, an antibacterial protein induced in the hemolymph ofSarcophaga peregrina (flesh fly) larvae,” J. Biol. Chem., vol. 260, pp.7174-7177 (1985). Although highly divergent from the cecropins anddefensins, the sarcotoxins presumably have a similar antibioticfunction.

Other lytic peptides have been found in amphibians. Gibson andcollaborators isolated two peptides from the African clawed frog,Xenopus laevis, peptides which they named PGS and Gly¹⁰Lys²²PGS. Gibsonet al., “Novel peptide fragments originating from PGL_(a) and thecaervlein and xenopsin precursors from Xenopus laevis,” J. Biol. Chem.,vol. 261, pp. 5341-5349 (1986); and Givannini et al., “Biosynthesis anddegradation of peptides derived from Xenopus laevis prohormones,”Biochem. J., vol. 243, pp. 113-120 (1987). Zasloff showed that theXenopus-derived peptides have antimicrobial activity, and renamed themmagainins. Zasloff, “Magainins, a class of antimicrobial peptides fromXenopus skin: isolation, characterization of two active forms, andpartial DNA sequence of a precursor,” Proc. Natl. Acad. Sci. USA, vol.84, pp. 3628-3632 (1987).

Synthesis of nonhomologous analogs of different classes of lyticpeptides has been reported to reveal that a positively charged,amphipathic sequence containing at least 20 amino acids appeared to be arequirement for lytic activity in some classes of peptides. Shiba etal., “Structure-activity relationship of Lepidopteran, a self-defensepeptide of Bombyx more,” Tetrahedron, vol. 44, No. 3, pp. 787-803(1988). Other work has shown that smaller peptides can also be lytic.See McLaughlin et al., cited below.

Cecropins have been shown to target pathogens or compromised cellsselectively, without affecting normal host cells. The synthetic lyticpeptide known as S-1 (or Shiva 1) has been shown to destroyintracellular Brucella abortus-, Trypanosoma cruzi-, Cryptosporidiumparvum-, and infectious bovine herpes virus I (IBR)-infected host cells,with little or no toxic effects on noninfected mammalian cells. SeeJaynes et al., “In vitro effect of lytic peptides on normal andtransformed mammalian cell lines,” Peptide Research, vol. 2, No. 2, pp.1-5 (1989); Wood et al., “Toxicity of a Novel Antimicrobial Agent toCattle and Hamster cells In vitro,” Proc. Ann. Amer. Soc. Anim. Sci.,Utah State University, Logan, Utah. J. Anim. Sci. (Suppl. 1), vol. 65,p. 380 (1987); Arrowood et al., “Hemolytic properties of lytic peptidesactive against the sporozoites of Cryptosporidium parvum,” J.Protozool., vol. 38, No. 6, pp. 161S-163S (1991); Arrowood et al., “Invitro activities of lytic peptides against the sporozoites ofCryptosporidium parvum,” Antimicrob. Agents Chemother., vol. 35, pp.224-227 (1991); and Reed et al., “Enhanced in vitro growth of murinefibroblast cells and preimplantation embryos cultured in mediumsupplemented with an amphipathic peptide,” Mol. Reprod. Devel., vol. 31,No. 2, pp. 106-113 (1992).

Morvan et al., “In vitro activity of the antimicrobial peptide magainin1 against Bonamia ostreae, the intrahemocytic parasite of the flatoyster Ostrea edulis,” Mol. Mar. Biol., vol. 3, pp. 327-333 (1994)reports the in vitro use of a magainin to selectively reduce theviability of the parasite Bonamia ostreae at doses that did not affectcells of the flat oyster Ostrea edulis.

Also of interest are the synthetic peptides disclosed in the followingpending patent applications, peptides that have lytic activity with asfew as 10-14 amino acid residues: McLaughlin et al., “AmphipathicPeptides,” U.S. patent application Ser. No. 08/681,075, filed Jul. 22,1996; and Mark L. McLaughlin et al., “Short Amphipathic Peptides withActivity against Bacteria and Intracellular Pathogens,” U.S. patentapplication Ser. No. 08/796,123, filed Feb. 6, 1997.

Lytic peptides such as are known generally in the art may be used inpracticing the present inventions. Selective toxicity toligand-activated cells is desirable, especially when the ligand andpeptide are administered separately. Selective toxicity is lessimportant when the ligand and peptide are linked to one another, becausein that case the peptide is effectively concentrated in the immediatevicinity of cells having receptors for the ligand.

Examples of such peptides are those designated D1A21 (SEQ. ID NO. 5),D2A21 (SEQ. ID NO. 6), D5C (SEQ. ID NO. 7), and D5C1 (SEQ. ID NO. 8).These peptides and other lytic peptides suitable for use in the presentinvention are disclosed in Jaynes, “Methods for the Design ofAmphipathic Peptides Having Enhanced Biological Activities,” U.S.provisional patent application Ser. No. 60/027,628, filed Oct. 4, 1996.In trials to date using these peptides alone (i.e., one of these fourpeptides without an associated ligand), in vitro LD₅₀ values againsthuman prostate cancer cell lines have ranged from about 0.57 μM to about1.61 μM. In trials to date using D2A21 alone (i.e., without anassociated ligand) LD₅₀ values against human breast, bladder, colon,cervix, lung, colon, and skin cancer cell lines have ranged from about0.28 μM to about 3.1 μM. For comparison, LD₅₀ has been measured to begreater than 100 μM for each of D2A21, D5C, and D5C1 for each of thefollowing types of normal, non-cancerous human cells: endothelial cells,fibroblasts, enteric cells, and keratinocytes. For D2A21, LD₅₀ has beenmeasured to be about 100 μM for human peripheral blood monocytes, and tobe greater than 100 μM for human peripheral blood T-cells.

Other GnRH analogs may be conjugated with a lytic peptide in accordancewith this invention. Among the analogs that may be used as part of sucha conjugate is I-LHRH-III (or I-GnRH-III), SEQ. ID NO. 16. This peptidehas been reported to suppress growth of several cancer cells. See I.Mezö et al., “Synthesis of Gonadotropin-Releasing Hormone III Analogs.Structure-Antitumor Activity Relationships,” J. Med. Chem. vol. 40, pp.3353-3358 (1997). The same I-LHRH-III selectively causes the release ofFSH. See W. Yu et al., “A hypothalamic follicle-stimulatinghormone-releasing decapeptide in the rat,” Proc. Natl. Acad. Sci USA,vol. 94, pp. 9499-9503 (1997); and U.S. patent application Ser. No.08/869,153, filed Jun. 4, 1997. Lytic peptide conjugates of I-LHRH-IIIwill be useful as contraceptives, and in the treatment of cancers suchas prostate cancers. Agonists of I-LHRH-III, such as are disclosed inU.S. patent application Ser. No. 08/869,153, may be used as well.

Miscellaneous

As used in the Claims, an “effective amount” of a composition is anamount sufficient to selectively kill the targeted cells in a backgroundpopulation of non-targeted cells. Where appropriate in context, an“effective amount” of a composition is also an amount that is sufficientto induce long-term contraception or sterility in an animal. Whereappropriate in context, an “effective amount” of GnRH or I-LHRH-III isan amount sufficient to temporarily restore fertility in an animal thathas been made sterile by destruction of gonadotropic cells. As used inthe Claims, the term “animal” is intended to include both human andnon-human metazoans.

The complete disclosures of all references cited in this specificationare hereby incorporated by reference; as are the full disclosures ofU.S. provisional application 60/041,009, filed Mar. 27, 1997; U.S.provisional application 60/057,456, filed Sep. 3, 1997; and of U.S.non-provisional application Ser. No. 08/869,153, filed Jun. 4, 1997. Inthe event of an otherwise irreconcilable conflict, however, the presentspecification shall control.

1. A method for killing or inhibiting the growth of agonadotropin-releasing hormone, lamprey III luteinizing hormonereleasing hormone (1-LHRH-III), β-subunit of luteinizing hormone (bLH),estrogen, testosterone, luteinizing hormone, chorionic gonadatropin,follicle stimulating hormone, melanocyte stimulating hormone, estradiol,dopamine, or somatostatin receptor expressing prostate, breast, ovarian,pituitary or endometrial tumor cell in a mammal, comprisingadministering to the mammal an effective amount of agonadotropin-releasing hormone, 1-LHRH-III, bLH, estrogen, testosterone,luteinizing hormone, chorionic gonadatropin, follicle stimulatinghormone, melanocyte stimulating hormone, estradiol, dopamine,somatostatin, or an analog of these hormones that binds to the receptorexpressing prostate, breast, ovarian, pituitary or endometrial tumorcell, and an effective amount of a lytic peptide, wherein said ligand islinked to said lytic peptide, and wherein said lytic peptide consists of10 to 39 amino acid residues, is basic, and forms an amphipathic alphahelix.
 2. A method as recited in claim 1, wherein the cell is part of apituitary adenoma, and wherein the hormone comprisesgonadotropin-releasing hormone, or lamprey III luteinizing hormonereleasing hormone (1-LHRH-III).
 3. A method as recited in claim 1,wherein the cell is part of a breast cancer, and wherein the hormonecomprises gonadotropin-releasing hormone or 1-LHRH-III.
 4. A method asrecited in claim 1, wherein the cell is part of an ovarian cancer, andwherein the hormone comprises gonadotropin-releasing hormone,1-LHRH-III, or β-subunit of luteinizing hormone (bLH).
 5. A method asrecited in claim 1, wherein the cell is part of a prostate cancer, andwherein the hormone comprises gonadotropin-releasing hormone or1-LHRH-III.
 6. A method as recited in claim 1, wherein the cell is partof an ovarian cancer, and wherein the hormone comprises 1-LHRH-III.
 7. Amethod as recited in claim 1, wherein the cell is part of a prostaticcancer, and wherein the hormone comprises 1-LHRH-III.
 8. A method asrecited in claim 1, wherein the cell is part of a breast cancer, andwherein the ligate hormone comprises 1-LHRH-III.
 9. A method as recitedin claim 1, wherein the cell is part of an endometrial cancer, andwherein the hormone comprises 1-LHRH-III.
 10. A method as recited inclaim 1, wherein the cell is part of a testicular cancer, and whereinthe hormone comprises gonadotropin-releasing hormone, 1-LHRH-III, orbLH.
 11. A method as recited in claim 1, wherein said hormone or hormoneanalog is bonded directly to said lytic peptide.
 12. A method as recitedin claim 1, wherein said lytic peptide is selected from the groupconsisting of a cecropin peptide, a melittin peptide, a defensinpeptide, a magainin peptide, a sarcotoxin peptide, and analogs of saidpeptides.
 13. A method as recited in claim 1, wherein said lytic peptidecomprises hecate.
 14. A method as recited in claim 1, wherein saidligand hormone, hormone analog, or said lytic peptide, or both compriseD-conformation amino acid residues.
 15. A method for killing orinhibiting the growth of a gonadotropin-releasing hormone, lamprey IIIluteinizing hormone releasing hormone (1-LHRH-III), β-subunit ofluteinizing hormone (bLH), estrogen, testosterone, luteinizing hormone,chorionic gonadatropin, follicle stimulating hormone, melanocytestimulating hormone, estradiol, dopamine, or somatostatin hormonereceptor expressing tumor cell in a mammal, comprising administering tothe mammal an effective amount of a hormone selected from the groupconsisting of gonadotropin-releasing hormone, 1-LHRH-III, bLH, estrogen,testosterone, luteinizing hormone, chorionic gonadatropin, folliclestimulating hormone, melanocyte stimulating hormone, estradiol,dopamine, somatostatin, and analogs of these hormones, and a lyticpeptide domain, wherein said hormone or hormone analog is linked to saidlytic peptide domain, and wherein said lytic peptide domain consists of10 to 39 amino acid residues, is basic, and forms an amphipathic alphahelix.
 16. A method as recited in claim 15, wherein said hormone orhormone analog is bonded directly to said lytic peptide domain.
 17. Amethod as recited in claim 15, wherein said lytic peptide domain isselected from the group consisting of a cecropin peptide, a melittinpeptide, a defensin peptide, a magainin peptide, a sarcotoxin peptide,and analogs of said peptides.
 18. A method as recited in claim 15,wherein said lytic peptide domain comprises hecate.
 19. A method asrecited in claim 15, wherein said hormone, hormone analog, or said lyticpeptide domain, or both comprise D-conformation amino acid residues.